This PPG has shown that certain clinical manifestations of Marfan Syndrome (MFS), caused by mutations in fibrillin-1, are mediated by high levels of active TGF-B and that progression of selected phenotypes is blocked by the angiotensin type 1 (ATI) receptor antagonist losartan. Work from Project 2 (Rifkin) has shown that latent TGF-S is activated in cultures of MFS vascular smooth muscle cells (VSMCs), that the activator of latent TGF-B is an MMP, most likely MMP-9, and that TGF-B stimulates AT1 receptor expression. We propose a model in which defective matrix yields abnormal latent TGF-B sequestration followed by activation, the active TGF-B stimulates enhanced ATI receptor and MMP-9 expression, MMP-9 activates latent TGF-B, and ATI receptor signaling promotes continued TGF-B expression. Thus, a cycle of activation, enhanced expression, and activation is established. However, the initiating event in this cycle is unknown, as are some ofthe interrelationships. This grant addresses three questions concerning TGF-B, ATI receptor, and MMPs in MFS.
In Aim 1, we will test whether perturbing the matrix results in the cycle of TGF-B formation, ATI receptor expression up-regulation, and MMP-mediated latent TGF-B activation and if these changes are interrelated.
In Aim 2, we will use FACS to isolate and characterize cells of different lineages that contribute to aortic root VSMC populations. Cells include cardiac nural crest, secondary heart field, mesoderm, and endothelium. Thus, we will determine whether or not abnormal cells in MFS arise from specific lineages, in which cells normally activate latent TGF-B, and have high levels of ATI receptor and MMPs. These results will be compared to those of Aim 1 in which cells generate these molecules because of failed matrix.
In Aim 3, we will generate MFS mice that are missing MMP-9 to establish if the in vitro activator MMP-9 is an in vivo activator. The completion of these aims will inform us as to the initiator of latent TGF-B activation, the cell that activates, and the nature ofthe in vivo activator

Public Health Relevance

of this work is that an understanding the fundamental cause of cellular phenotypes in Marfan Syndrome will allow us to develop novel therapeutic approaches. We, the PPG, demonstrated this possibility in the last grant cycle. In this new proposal, we will attempt to identify additional targets, as it is probable that different people or different tissues with MFS will require additional therapies.